+ the specification of software on our PC +
The Windows system

The applications
- ZEN Black: ZEISS microscope application to operate the microscope for image acquisition and other operations
- imgeJ/Fiji: open source application for mask generation
- Spyder: Integrated development environment (IDE) for python code
- Sikulix: automation platform in Windows environment
- Google Chrome: the web browser where the Gmail web-portal is opened and called by Sikulix to send warning message

Warning
Directly use our Sikulix codes on a computer other than ours will fail with no doubt for many reasons. Please refers to “Insstruction of Sikulix code.html”.
step-by-step tutorial
1 prepare sample slide
prepare the cell or tissue culture on a coverslip.
mount it with 1 μM biotin-benzophenone in 50:50 DMSO:water and store in dark.
the biotin-benzophenone is photo-active under two-photon excitation at 720 nm
2 photo-labeling on microscope
2.1 load the slide on microscope (we use zeiss LSM 880) and use 25x oil immersion lens.
2.2 start the applications on Windows:
2.3 setup the tiles
the workflow
please refer to the video
2.3.1 find the boundary points along the coverslip.
move along the edge of the coverslip. Stop at a location and manually focus on the sample.
add the location to the ‘Positions’ Panel.
keep going until 6~8 locations are collected.
save the boundary position list.
2.3.2. calculate the tiles within the shape refined in the boundary points.
call the script “tileScanConvexHullz_split.py” in Spyder
line 12: enter the path of the pre-saved “.pos” file of the boundary list
line 14: enter value of the tile size, always NOT smaller than of the scale of field of view. i.e. 25x lens on our LSM 880, the size of each field is 340.1 μm.
line 16: the maximum numbers of tiles can be written into a position file.
ZEN Black may freeze when a positioin file contains more than 120 positions is loaded.
run the script “tileScanConvexHullz_split.py” after the parameters are entered.
position files are generated: “tilePos-1.pos”, “tilePos-2.pos”, “tilePos-3.pos”,…
the position file is loaded into Zen Black in the “Positions” panel

2.3.3. setup the imaging parameters
load a position file, move to a tile position, then tune the laser powevr, receiver gain, and etc. to get an image with decent signal.
test 1~2 tiles. Then the imaging parameters are settled.
warning: close all active images in Zen Black after the test.
2.3.4. Mask generation in imageJ/Fiji
install the Fiji macro used for mask generation
“self.ijm” and “donut.ijm” are provided

For instance, the macro functions are shown as below after “donut.ijm” is loaded

test the processing of images acquired in step 2.3.3. Make sure the ijm macro works properly.
Warning: after test, close all active images and windows except the console of imageJ/Fiji by calling the macro, clearWindows.
2.4 automation with the Sikulix code
3 count the pixels STOMPed to estimate the amount of tagged proteins.
Note 3.1: the images and log files are organized into the folders by the order of the position files generated at the step 2.3.2.
3.1. run the python code “TotalPixels_allLogs.py” in Spyder.
line 15: enter the path of the parent folder generated at step 2.4.1.

the script will iterate through all its subfolder and collect and summarize the total pixels.

Note 3.2: Typically, labeling 4 - 5 million pixels with mask resoution: 512x512 pixel-by-pixel under 25x lens should render sufficient proteins for one sample for Mass Spec submission.
Approximately four days per sample on the microscope is required to label adequate proteins.
4 Lift the STOMPed coverslip and store it
4.1 unload the slide from the microscope once the STOMPing is finished. Clean the immersion oil With a cotton swab, then soak the slide in the pure water for more than half an hour on a shaker.
4.2 gently peel off the nail polish seal around the coverslip, and transfer the coverslip to a parafilm with the sample side up.
4.3 clean the free affinity tag off the coverslip
add 1 mL 50:50 DMSO:water to the coverslip, and asperate the liquid after 5 min. Repeat this 3 times.
add 1 mL water to the coverslip, and asperate the liquid after 5 min. Repeat this 3 times.